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Journal of General Virology 2006
Core Protein Cleavage by Signal Peptide Peptidase is Required for
Hepatitis C Virus-Like Particle Assembly
Malika Ait-Goughoulte, Christophe Hourioux, Romuald Patient, Sylvie Trassard,
Denys Brand, and Philippe Roingeard.
INSERM ERI 19, Université François Rabelais, Faculté de Médecine & CHU, 10 boulevard
Tonnelle, 37032 Tours, France.
Running title: HCV core protein cleavage by SPP and viral assembly
Correspondence: Philippe Roingeard, Laboratoire de Biologie Cellulaire, Faculté de
Médecine de Tours, 10 boulevard Tonnellé, F-37032 Tours Cedex France.
Tel (33) 2 47 36 60 71
Fax (33) 2 47 36 60 90
E-mail: [email protected]
Additional footnote: M.A.-G. and C.H. contributed equally to this work.
SUMMARY
Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon,
self-assembles into HCV-like particles (HCV-LP) at the endoplasmic reticulum (ER)
membrane, providing an opportunity to study HCV assembly and morphogenesis by electron
microscopy. We used this model to investigate whether the processing of the HCV core
protein by the signal peptide peptidase (SPP) is required for the HCV-LP assembly. We
designed several mutants as there are conflicting reports concerning the cleavage of mutant
proteins by SPP. Production of the only core mutant protein that escaped SPP processing led
to the formation of multiple layers of electron-dense ER membrane, with no evidence of
HCV-LP assembly. Our data shed light on the HCV core residues involved in SPP cleavage
and suggest that this cleavage is essential for HCV assembly.
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The hepatitis C virus (HCV) genome contains approximately 9,600 nucleotides. At both the
5’ and 3’ ends, untranslated regions flank a single open reading frame encoding a single
polyprotein precursor of about 3,000 amino acids (aa). This precursor is co- and posttranslationally processed by cellular and viral proteases, to yield three mature structural
proteins [one core (C) and two envelope (E1 and E2) proteins] and six non-structural proteins
involved in polyprotein processing and viral RNA replication (Grakoui et al., 1993). The
hydrophobic sequence at the junction between the HCV core protein and the envelope
glycoprotein E1 (amino acids 170 to 191 in the polyprotein) functions as a signal sequence
and targets the nascent polyprotein to the ER membrane, inducing the translocation of E1 into
the lumen of the ER (Lo et al., 1995). Cleavage by a signal peptidase in the ER lumen
releases the N-terminal end of E1, leaving the 191-aa core protein anchored by the signal
peptide (McLauchlan 2000). This 191-aa polypeptide (p23) is an immature form of the core
protein, and is further processed by an intramembrane presenilin-type aspartic protease SPP
(for signal peptide peptidase) (Weihofen et al., 2002). The site of the SPP cleavage is unclear
but probably lies between residues 173 and 179, resulting in the elimination from the mature
core protein of all or part of the N-terminal hydrophobic domain containing the signal peptide
of E1 (McLauchlan et al., 2002). The resulting mature HCV core protein (p21) is thought to
constitute the HCV capsid and therefore to be an integral component of the virion. Indeed,
this mature form of the HCV core protein is the predominant form detected in virus particles
purified from the sera of patients with chronic HCV infection (Yasui et al., 1998). However,
processing of the HCV core protein by SPP has also been shown to release the core protein
from the ER membrane leading to its trafficking to zones of the ER in which lipid droplets are
produced (McLauchlan et al., 2002). During its trafficking, the proline-rich, central
hydrophobic domain 2 of the HCV core protein (amino acids 119 to 173) is though to remain
anchored in the cytoplasmic phospholipid monolayer of the ER, and is therefore thought to be
present in the membrane surrounding the lipid droplets, which consists of a single
phospholipid monolayer (Hope & McLauchlan, 2000).
Thus, retention of the HCV core protein in the ER membrane by its sequence signal may play
an essential role in viral particle assembly by, at least transiently, preventing trafficking to the
lipid droplets. It therefore remains unclear whether HCV core protein cleavage by SPP is an
essential step for virion assembly and morphogenesis. Recent advances in the establishment
of cell culture systems have made it possible to produce infectious HCV that can be
efficiently propagated in cultured, naive hepatoma cells (Lindenbach et al., 2005; Wakita et
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al., 2005; Zhong et al., 2005). However, it remains extremely difficult to observe virus
assembly and morphogenesis in these cell cultures (P.R., personal observations). We have
developed a model based on Semliki forest virus (SFV) replicon vectors, in which the
production of the HCV core protein in mammalian cells leads to the assembly of this protein
into HCV-like particles (HCV-LP) (Blanchard et al., 2003). This model displays abortive
HCV-LP budding, but it remains a useful tool for studies of the early events of HCV
assembly in eukaryotic cells (Roingeard et al., 2004). In this study, we use this HCV capsid
assembly model to characterise the role of SPP cleavage in viral morphogenesis.
HCV core mutants. Previous studies have identified various residues in the transmembrane
signal sequence domain of the HCV core protein that are involved in the intramembrane
cleavage of this protein by SPP. It was initially demonstrated that the ASC180/3/4VLV HCV
mutant core protein, which contains helix-favouring residues in place of the helix-bending
and helix-breaking residues, was not processed by SPP (Lemberg & Martoglio, 2002;
McLauchlan et al., 2002). However, a recent study of the same ASC180/3/4VLV mutant
demonstrated efficient cleavage of this molecule by SPP (Okamoto et al., 2004). In the same
study, it was shown that an IF176/7AL core mutant protein was not processed by SPP. These
discrepancies were observed for the core proteins of both the 1a and 1b genotypes of HCV.
They were not due to differences in the core protein sequence or in cell type, as both studies
were conducted with BHK-21 cells. We therefore designed SFV replicon vectors encoding
the ASC180/3/4VLV and IF176/7AL core mutants, to resolve these discrepancies and to
address our own research questions. The SF173/4ML mutant core protein was found not to be
processed by SPP in a third study (Yamanaka et al., 2002), and was therefore also
investigated in our study. The C191 and C173 sequences were subcloned in SFV vectors from
the HCV cDNA Dj 6.4 clone (genotype 1a) as previously described (Blanchard et al., 2003).
Site-directed mutagenesis within the C-terminal C191 signal sequence was performed with
antisense primers leading to (i) generation of the three mutants ASC180/3/4VLV, IF176/7AL
and SF173/4ML (Fig. 1A) and (ii) insertion of a stop codon at the 3’ end of the core proteincoding region.
Cleavage of HCV wild-type and mutant core proteins by SPP. The BHK 21 cell line was
cultured and electroporated as previously described (Blanchard et al., 2002). SDS-PAGE and
western blotting analysis showed that the wild-type (WT) C191 core protein was fully cleaved
by SPP in these cells, as previously reported (Blanchard et al., 2003) (Fig. 1B). In cells
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transfected with the WT C191 construct and treated with various concentrations (60 µM, 100
µM or 150 µM) of (Z-LL)2-ketone (Calbiochem), SPP cleavage was partially inhibited (Fig.
1B), as reported in previous studies using this commercially available SPP inhibitor
(Weihofen et al., 2003). We used Image J gel analyser software to determine the amounts of
p23 and p21 forms of HCV core protein in the scanned blots. Uncleaved and cleaved forms
were detected in equal quantities in cells treated with 60 M of (Z-LL)2-ketone. Similar
quantities of uncleaved (60%) and cleaved (40%) forms were detected at concentrations of
100 M and 150 M of (Z-LL)2-ketone, indicating that this protease inhibitor was most
efficient at a concentration of 100 M (Fig. 1B). This is not surprising, as this compound is
very efficient in cell-free translation/translocation assays, but less efficient in cellular assays
because it does not readily cross the plasma membrane (Weihofen et al., 2003).
The ASC180/3/4VLV mutant core protein was the only mutant protein studied that remained
uncleaved, resulting in detection of the p23 form of the core protein alone (Fig. 1B). This
confirms the results obtained by Martoglio’s group with this mutant (Lemberg & Martoglio,
2002; McLauchlan et al., 2002), but conflicts with the data reported by Okamoto et al. (2004).
We found that the IF176/7AL mutant core protein was efficiently processed by SPP, leading
to detection of the p21 core protein (Fig. 1B), despite a previous report of this protein being
resistant to SPP cleavage (Okamoto et al., 2004). The SF173/4ML mutant core protein was
also efficiently cleaved in our study (Fig. 1B). A total lack of processing (Yamanaka et al.,
2002), or partial processing (Liu et al., 1997) had previously been reported for this protein.
However, it should be pointed out that mutations in the HCV core protein have been shown to
have a significant effect on the mobility of this protein in SDS-PAGE (McLauchlan et al.,
2002). In addition, we cannot rule out the possibility that SPP processes the signal sequence
of the core protein heterogeneously, at several sites, as observed for -secretase, an aspartic
protease closely related to SPP that cleaves the transmembrane region of the amyloid-
precursor protein at several sites (Edbauer et al., 2003). These factors may account for
difficulties in interpreting SDS-PAGE results for HCV mutant core proteins, and highlight the
need for comparisons with relevant reference peptides. It should be also noted that the
interaction of the HCV mutant core proteins with lipid droplets was not investigated in most
of these previous studies. This led us to investigate the subcellular distribution of our HCV
mutant core proteins.
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Subcellular distribution of the HCV wild-type and mutant core proteins. This analysis
was made with the WT and mutant constructs expressed in the human hepatoma FLC4 cell
line (Aizaki et al., 2003) (Fig. 1C). Expression from SFV vectors was found to be less
efficient in this cell line than in BHK-21 cells, allowing a more precise analysis of the
subcellular core distribution and lipid droplet interaction. All the core proteins except the
ASC180/3/4VLV mutant core protein were colocalized with lipid droplets (Fig. 1C). Nile red
labeling in control cells transfected with -Gal SFV RNA showed that lipids were evenly
distributed throughout the cytoplasm. Transfection with the HCV WT C191 and C173
construct induced the clustering in the perinuclear area of large lipid droplets staining strongly
for HCV core protein (Fig. 1C). The IF176/7AL and SF173/4MLmutant core proteins had a
subcellular distribution similar to that of WT C191 (Fig. 1C). The ASC180/3/4VLV mutant
core protein was weakly associated with some lipid droplets, but was mostly distributed in
reticular patches throughout the cytoplasm (Fig. 1C). Thus, all the HCV core mutant proteins
cleaved by SPP in SDS-PAGE analysis were colocalized with lipid droplets, like the WT core
protein. This confirmed that the ASC180/3/4VLV mutant core protein was the only mutant
core protein that escaped SPP processing.
Ultrastructural changes induced by the HCV wild-type and mutant core proteins. This
study is the first to report the ultrastructural changes induced in cells by production of the
HCV mutant core proteins ASC180/3/4VLV, IF176/7AL and SF173/4ML. BHK-21 cells
transfected with the recombinant SFV RNAs encoding the two mutant HCV core proteins
correctly cleaved by SPP (IF176/7AL and SF173/4ML) differed markedly in terms of
ultrastucture (Fig. 2). As expected, the SF173/4ML mutant core protein gave a pattern similar
to that of WT C191, with convoluted ER membranes displaying HCV-LP assembly (Fig. 2).
Conversely, cells producing the IF176/7AL mutant core protein showed no sign of ER
modification (Fig. 2), like cells transfected with -Gal RNA or C173 RNA. This provides
additional evidence that this mutant core protein is efficiently cleaved by SPP, and does not
remain anchored in the ER membrane by its transmembrane sequence signal. This finding
may also suggest that amino acids 176 and 177 (IF in the WT core protein) are located in the
mature core protein after SPP cleavage and their replacement by other amino acids may
hamper core protein multimerization.
Cells transfected with the recombinant SFV RNA encoding the ASC180/3/4VLV mutant
HCV core protein, which is not processed by SPP, had a very unusual ultrastructure. Indeed,
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convoluted electron-dense ER membranes were found to form multiple layers in these cells
(Fig. 3). No HCV-LPs or marked clustering of lipid droplets was observed in cells producing
this mutant core protein. These observations confirm that the ASC180/3/4VLV core protein
remains anchored in the ER membrane by its transmembrane sequence signal. This
unprocessed mutant protein therefore probably displays self-interaction, inducing the
formation of multi-layer ER membranes, but not assembly to give HCV-LP. These data
suggest that processing of the HCV core protein by SPP is required for assembly of the virus
particle. We investigated the ultrastuctural changes induced in cells by production of the WT
C191 core protein in the presence of (Z-LL)2–ketone. Unfortunately, this commercially
available SPP inhibitor is not very efficient in cellular assays, due to low plasma membrane
permeability (Weihofen et al., 2003). We were able to achieve only 60% inhibition of SPP in
cells transfected with the WT C191 core protein construct and treated with 100 m
concentration (Z-LL)2–ketone. This treatment decreased the frequency of HCV-LP, which
became much harder to detect, and led to the generation of multi-layer ER membranes similar
to those found in cells producing the ASC180/3/4VLV mutant core protein (data not shown).
Thus, cleavage of the transmembrane signal sequence of the HCV core protein by SPP
appears to be an essential step in assembly of the viral particle. This conclusion is at odds
with the recent results of analyses of WT C191 production in yeast cells (Majeau et al., 2005).
The reasons for these discrepancies are unknown but may be due to differences in the HCV
assembly mechanisms in yeast and mammalian cells. However, the intracellular HCV-LP
assembly has not been well established by EM in yeast cell sections.
In conclusion, HCV core protein processing by SPP is required for the trafficking of the
protein to the lipid droplets, but we suggest that it is also required for assembly into virus
particles at the ER membrane. Our data provide new insight into the relevance of interactions
between HCV and host cell factors, and suggest that SPP inhibitors are of potential value for
the treatment of HCV infection.
ACKNOWLEDGEMENTS
We thank Mario Mondelli and Yoshiharu Matsuura for providing us with the monoclonal
human anti-core B12F8 and the FLC4 cell line, respectively. This work was supported by a
grant from the French National Agency for Research on AIDS and Viral Hepatitis (ANRS),
and by the “Région Centre”. M.A.-G. and R.P. were supported by fellowships provided by the
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French Ministry of Research. M.A.-G. also received a grant from the French Foundation for
Medical Research (FRM). Our data were generated with the help of the RIO Electron
Microscopy Facility of the François Rabelais University of Tours.
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Fig. 1. Analysis of HCV core-E1 signal peptide processing. (a) Signal sequences of WT C191
HCV core protein and mutants (IF176/7AL, ASC180/3/4VLV and SF173/4ML). The
transmembrane region of the signal sequence is underlined. (b): Production in BHK-21 cells
of the WT and mutant C191 proteins and analysis of their processing by SDS-PAGE and
western blotting, using the human monoclonal anti-HCV core antibody B12F8 (Esposito et
al., 1995). The mutants were compared with the WT C191 protein in the absence or presence
of various concentrations of the SPP inhibitor (Z-LL)2–ketone, and with the reference core
protein C173. (c). Immunofluorescence of WT and mutant HCV core proteins combined with
Nile Red staining of lipid droplets in FLC4 cells.
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Fig. 2. Electron micrographs of BHK-21 cells electroporated with WT C191 (a & b), C191
SF173/4ML (c & d), or C191 IF176/7AL (e & f) RNA. Micrograph in a shows a zone of
convoluted ER membranes (delimited by arrowheads) in which HCV-LP assembly (arrows)
was frequent. Micrograph in b shows that clusters of large lipid droplets were found in the
perinuclear area next to ER membranes in which HCV-LP assembly (large black arrows) was
detected. Cells transfected with C191 SF173/4ML RNA presented a pattern similar to that of
the WT protein, with convoluted ER membranes (delimited by arrowheads in c) in which
HCV-LP assembly (arrows in d) was detected. Cells transfected with C191 IF176/7AL RNA
had normal ER structures, evenly distributed throughout the cytoplasm (white arrows), in
which no HCV-LP was detected, as for cells transfected with -Gal recombinant RNA (not
shown). The only subtle change in ultrastructure of cells producing C191 IF176/7AL was the
clustering of lipid droplets in the perinuclear area, next to normal ER membranes (micrograph
in f). N, nucleus. ld: lipid droplet. ER lu: ER lumen. Bars in a and d, 0,2 m. Bars in b, c, e
and f, 1 m.
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Fig. 3. Electron micrographs of BHK-21 cells electroporated with the C191 ASC180/3/4VLV
RNA, showing electron-dense ER membranes (delimited by arrowheads at low magnification
in a). At high magnification (c), these structures were found to be formed by the interaction of
multiple ER membranes (arrow). In b, an immunogold labeling with anti-core antibodies
demonstrated the presence of high amount of core protein in these multi-layered ER
membranes. m. Bars in b and c, 100 nm.